Identification Of Gene Targets For Live Bacterial Vaccine Development

Immuno-prophylaxis can play a significant role in prevention of bacterial diseases like haemorrhagic septicaemia (HS), black quarter (BQ) etc. In fact vaccines are available and in use against many bacterial diseases. These current vaccines are formulation of inactivated whole cell, i. e. killed bacteria. These traditional vaccines are prepared by growing and inactivating the bacterial cultures. Although these vaccines are very safe in use but during the process of inactivation many bacterial antigens including surface exposed proteins (which are highly immunogenic in nature) get denatured and damaged. Therefore the major drawbacks of killed vaccines use are 1) they are not capable to induce robust immune response 2) the induced immunity in short lived. Therefore multiple doses of the vaccines are required and boosters are needed very frequently

i. e. at an interval of 6–12 months period. Alternative to killed, live attenuated vaccines can be developed by removing the virulence associated gene(s) or part of the gene(s) from the bacteria. After gene inactivation the bacterium remains live but unable to cause disease i. e. attenuated. Therefore live attenuated vaccines mimic the natural infection without producing the disease. The animal body see them as natural infection and mount a robust immune response, thus the generated immunity is sufficient to protect animals for several years to lifetime. More over only single or couple of doses are required for vaccination. The key behind the development of live vaccine is the identification of virulence genes in bacteria. There are several methods to identify virulence associated genes in bacteria. The transposon mediated random mutagenesis is a simple and effective way to accomplish the task. Transposon contains two parts that includes mosaic (ME) ends and an antibiotic selection marker driven by promoter of constitutively expressed gene. The mosaic ends can get incorporated in the bacterial genome randomly thus inactivates the gene function. Ideally transposon hits randomly and once per genome therefore covers almost all genes of the target bacteria.

Steps for identification of targets for live vaccine development using transposon mediated mutagenesis are as follows:

Construction of transposon in transposon construction vector.

Make bacterial culture electrocompetent and electroporate the transposon to bacteria.

Select the transformants on antibiotic selection media.

Assembly of mutant bank: pick isolated colonies, grow and freeze them at −80°C after addition of glycerol. The mutant bank will be having mutants where in each colony a unique gene is disrupted. The library should be over saturating means number colonies to be picked should be double than the number of genes in genome.

The each colony should be tested for some in vitro assay like phagocyte killing assays to identify attenuated gene mutant(s).

The identity of disrupted gene can be known by sequencing of isolated genomic DNA using outward facing primers in the transposon sequences and BLAST search.

Now these mutants can be used to test in laboratory animals followed by target species of animal for their attenuation and vaccine potential.